组蛋白去乙酰化酶抑制剂SAHA联合伊马替尼对K562细胞的协同杀伤作用

目的:观察组蛋白去乙酰化酶( HDAC)抑制剂SAHA联合伊马替尼(imatinib,IM)对人慢性髓系白血病(CML)细胞株的协同杀伤作用,并探讨其分子学机制.方法:将不同浓度的SAHA和IM分别或联合作用于对数生长期的K562细胞,MTT比色法检测药物对细胞的生长抑制作用；应用Hoechst荧光染色法和流式细胞仪(FACS)检测细胞凋亡,Western blot法检测药物处理后Bcr-Abl及其下游信号途径蛋白的表达、Caspase途径活化和凋亡相关蛋白表达的改变.结果:SAHA和IM单独作用K562细胞呈剂量依赖性抑制细胞生长(SAHA:F=433.8,P＜0.001；IM:F =54.14,P＜0.001)；两药联合对细胞的生长有协同杀伤作用,联合指数CI＜1.FACS分析和Hoechst荧光染色证实,SAHA和IM两药联合能加速K562细胞的凋亡,激活Caspase-3、Caspase-8和PARP,下调抗凋亡蛋白Mcl-1表达.此外,SAHA和IM联用能抑制Bcr-Abl及其磷酸化水平,下调JAK2和磷酸化STAT5表达.结论:HDAC抑制剂SAHA联合IM能协同杀伤CML细胞、加速细胞凋亡.其作用机制可能是两药联用协同抑制了Bcr-Abl及其磷酸化水平,抑制JAK2/STAT5信号途径和下调下游靶基因Mcl-1.

Synergistic effect of histone deacetylase inhibitor suberoylanilide hydroxamic acid with imatinib on K562 cells

Objective: To investigate synergistically killing effect of histone deacetylase(HDAC) inhibitor suberoylanilide hydroxamic acid(SAHA) combined with imatinib on human chronic myeloid leukemia(CML) cell line.Methods: K562 cells were co-treated with SAHA and imatinib.Cell growth was measured by MTT assay.Apoptosis was determined using Hoechst staining apoptosis detection kit and flow cytometric analysis.Activation of Caspase pathway,expression of Bcr-Abl and its downstream target genes,and expression of anti-apoptotic proteins were detected by Western blot.Results: SAHA synergized the cytotoxicity of imatinib against leukemia K562 cells,concomitantly with increased apoptosis and enhanced activation of Caspase-3,-8 and PRAP.The combination therapy resulted in significantly lower levels of Bcr-Abl,phosphorylated Bcr-Abl compared to treatment with either SAHA or imatinib alone.Furthermore,the co-treatment resulted in down-regulation of anti-apoptotic protein Mcl-1 expression.Also,marked down-regulated expression of JAK2,STAT5,and phosphorylated STAT5 was detected in the combination therapy.Conclusions: Combining HDAC inhibitor SAHA with imatinib can kill CML cells synergistically by inhibiting cell growth and inducing apoptosis,which is associated with activation of Caspase pathway and regulation of anti-apoptotic proteins.